University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Doctoral Dissertations Graduate School 12-2007 Gene Expression Analysis of Escherichia coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays Kristina K. Carter University of Tennessee - Knoxville Follow this and additional works at: https://trace.tennessee.edu/utk_graddiss Part of the Food Science Commons Recommended Citation Carter, Kristina K., "Gene Expression Analysis of Escherichia coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays. " PhD diss., University of Tennessee, 2007. https://trace.tennessee.edu/utk_graddiss/134 This Dissertation is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a dissertation written by Kristina K. Carter entitled "Gene Expression Analysis of Escherichia coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays." I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Food Science and Technology. David A. Golden, Major Professor We have read this dissertation and recommend its acceptance: Michael Davidson, Doris D’Souza, Todd Reynolds, Arnold Saxton Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) To the Graduate Council: I am submitting herewith a dissertation written by Kristina K. Carter entitled “Gene Expression Analysis of Escherichia coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays.” I have examined the final electronic copy of this dissertation for form and content and recommend that it be accepted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, with a major in Food Science and Technology. David A Golden, Major Professor We have read this dissertation and recommend its acceptance: Michael Davidson Doris D’Souza Todd Reynolds Arnold Saxton Accepted for the Council: Carolyn R. Hodges Vice Provost and Dean of the Graduate School (Original signatures are on file with official student records.) Gene Expression Analysis of Escherichia coli O157:H7 at 10 and 37°C and Under Acidic Conditions Using High Density Oligonucleotide Microarrays A Dissertation Presented for the Doctor of Philosophy Degree The University of Tennessee, Knoxville Kristina K. Carter December, 2007 Copyright © 2007 Kristina K Carter All Rights Reserved ii Acknowledgements The author with gratitude and appreciation, wishes to acknowledge the contributions of all those who have given their time, attention, and expertise in order to make this achievement possible. To my committee members, thank you for your guidance and encouragement over the past three years. To the faculty and students of the UT Food Science and Technology Department, thank you for your support and encouragement. I greatly appreciate everything each of you has done for me. iii Abstract This purpose of this investigation was to use DNA microarrays to study gene expression in E. coli O157:H7 under refrigerated and acidic conditions. Total RNA from E. coli O157:H7 grown to 7 log CFU/mL under control (37 °C, pH 7), refrigerated (10°C, pH 7), acid adapted (37°C, pH 5.5, then lowered to 3.5), and acid shocked (37°C, pH 7, then lowered to 3.5) conditions was extracted using an optimized Qiagen RNeasy procedure. Total RNA was converted to cDNA, labeled, and hybridized onto an Affymetrix GeneChip® E. coli Genome 2.0 Array. Results were analyzed using Analysis of Variance (ANOVA) and a t-test with significance set at p<0.01 and an expression cut- off of 2-fold difference in gene expression. Expression of selected genes, including an internal control (hemX) was confirmed using real time reverse transcriptase PCR. Microarray results revealed 293 down-regulated and 375 up-regulated genes. Cold shock genes cspE, cspA, cspG, and cspH were down-regulated; recA and SOS DNA repair genes uvrB, yebG, ruvA and B, lexA, and dinl were up-regulated. Expression of fhuA (outer membrane protein) and napG (electron transport) genes was 115- and 70-fold greater under refrigeration. Microarray analysis revealed that genes involved in DNA binding, biosynthesis, iron, transport and cellular metabolism were significantly up-or down-regulated in acid adapted cells. Genes related to iron uptake and transport (cyo, ent, fhu, fep, and feo operons) were up-regulated under acid shocked conditions. For acid adapted conditions, expression of cfa (cyclopropane fatty acid biosynthesis; decreases membrane fluidity and increases acid resistance) and genes involved in the glutamate decarboxylase acid resistant systems were significantly up-regulated. The virulence genes eae, stx1, and stx2 were not differentially expressed under either condition. iv Table of Contents Chapter Page I. REVIEW OF CURRENT LITERATURE 1 Introduction 2 Escherichia coli O157:H7 3 History 3 Mechanisms of Infection 7 Detection Methods 10 Outbreaks 15 Apple (Malus domestica) Cider 17 Microarrays 24 Spotted Microarrays 26 High-Density Oligonucleotide Arrays 27 Comparison of Arrays Platforms 28 Application of Microarrays in Food Microbiology 31 Gene Expression Responses to Environmental Stresses 35 Background 35 Global Stress Regulator 36 Response to Acid Stress 38 Response to Cold Stress 45 Specific Aims 49 Hypothesis 49 Objectives 49 References 51 Appendix 60 II. OPTIMIZATION OF TOTAL RNA EXTRACTIONS FROM ESCHERICHIA COLI O157:H7 EDL 933 FOR MICROARRAY ANALYSIS 63 Abstract 64 Introduction 66 Materials and Methods 69 Cell Culture 69 Total RNA Extractions 69 Absorbency Measurements 71 Statistical Analysis 71 Results and Discussion 71 References 76 Appendix 77 III. COMPARISION OF GENE EXPRESSION IN ESCHERICHIA COLI O157:H7 EDL 933 AT 10 AND 37°C USING HIGH-DENSITY OGLIONUCLEOTIDE MICROARRAYS 82 Abstract 83 Introduction 84 Materials and Methods 86 Cell Cultures 86 v RNA Extractions 86 Absorbency Measurements 87 Microarrays 87 cDNA Synthesis and Labeling 88 Array Hybridization, Washing and Scanning 89 Statistical Analysis 89 Microarray Confirmation 90 Results and Discussion 91 Microarray 91 rt-RT-PCR confirmation 102 Conclusions 104 References 105 Appendix 111 IV. COMPARISION OF GENE EXPRESSION IN ESCHERICHIA COLI O157:H7 EDL 933 UNDER ACIDIC CONDITIONS USING HIGH-DENSITY OGLIONUCLEOTIDE MICROARRAYS 131 Abstract 132 Introduction 133 Materials and Methods 140 Cell Cultures 140 RNA Extractions 140 Absorbency Measurements 141 Microarrays 141 cDNA Synthesis and Labeling 142 Array Hybridization, Washing and Scanning 143 Statistical Analysis 143 Microarray Confirmation 144 Results and Discussion 145 Acid Adapted Microarrays 145 Acid Shocked Microarrays 145 Acid Adapted vs. Acid Shocked Microarray Study 160 rt-RT-PCR confirmation 162 Time Point Analysis 163 Conclusions 166 References 168 Appendix 176 VITA 239 vi List of Figures Figure Page 1.1 Chemical structure of malic acid (L-form) 61 1.2 Leaflet message produced by the US Apple Association and provided to consumers at apple cider retail outlets 62 2.1 Growth curve for E. coli O157:H7 EDL 933 incubated at 37°C (control) 77 2.2 Growth curve for E. coli O157:H7 EDL 933 incubated at 10°C 77 2.3 Growth curve for E. coli O157:H7 EDL 933 adapted to pH 5.5 78 2.4 Comparison of TRIzol® and Qiagen extraction protocols for purity of RNA 79 2.5 Comparison of Qiagen modification steps for acid adapted and acid shock cells 81 3.1 Plot of p-values vs. differential expression (fold change) of genes from E. coli O157:H7 control vs. refrigeration treatments 111 3.2 Hydroxamate bacterial sideophores 112 3.3 FhuA-TonB complex 112 3.4 Differences in expression level of refrigeration genes 115 3.5 Ct curves for hemX 116 3.6 Tm graph for hemX 117 3.7 Control and refrigeration hemX dilutions 118 3.8 Ct curves for napG 119 3.9 Tm curve for napG 120 3.10 Gel of napG dilutions 121 3.11 Ct curves for fhuA dilutions 122 3.12 Tm curve for fhuA 123 3.13 Gel of fhuA dilutions 124 3.14 Ct curves for ompT dilutions 125 3.15 Tm curve for ompT 126 3.16 Gel of ompT dilutions 127 3.17 Ct curves of gltJ dilutions 128 3.18 Tm curve of gltJ 129 3.19 Gel of gltJ dilutions 130 4.1 Plot of p-values vs. differential expression (fold change) of genes from E. coli O157:H7 control vs. acid adapted treatments 176 4.2 Plot of p-values vs. differential expression (fold change) of genes from E. coli O157:H7 control vs. acid shocked treatments 177 4.3 Structure of hydroxamate bacterial sideophores 178 4.4 FhuA-TonB complex 178 4.5 Plot of p-values vs. differential expression (fold change) of genes from E. coli O157:H7 acid shock vs. acid adapt treatments 179 4.6 Differential expression levels of acid adapted genes 182 4.7 Differential expression levels of acid shocked genes 183 4.8 Differential expression levels of gadB at 0, 5, 15 and 60 minutes 184 4.9 Differential expression levels of gltJ at 0, 5, 15 and 60 minutes 185 4.10 Ct curves for hemX 186 4.11 Tm graph for hemX 187 4.12 Gel of hemX dilutions 188 vii 4.13 Acid adapted hemX Ct curves for time